DNA TOPOISOMERASES--DNA INTERACTIONS AND AT CONTROL

我的研究的长期目标是理解 线粒体生物发生在基因水平的详细机制 酶的结构和功能以及线粒体DNA水平 组织和表达。这项提案的具体目标是 为了开始阐明DNA拓扑异构酶在 线粒体DNA的复制和代谢。尤其是， 三磷酸腺苷与核和线粒体I型相互作用的研究 将对拓扑异构酶进行研究。事实证明， 不依赖于ATP的I型拓扑异构酶实际上是被 三磷酸腺苷。因为这种效应已经从人类的TOPO中观察到 白血病、HeLa和小牛胸腺细胞，我们假设 ATP对Topo I活性的调节可能是一种普遍现象 哺乳动物细胞。核苷酸酶的细节 以及这一规则的相互作用和机制 体内和体外研究将被阐明。 这些酶在线粒体DNA复制和转录中的作用 新陈代谢的定义将通过研究 拓扑异构酶I和II与体内线粒体基因组和 在试管中。这些研究将使用几种抗肿瘤药物， 已经被证明能特异性地干扰拓扑异构酶- 催化的断链和重新连接过程，使得在 蛋白质变性剂的存在，DNA裂解会导致 DNA末端共价连接的拓扑异构酶 碎片。特别是，喜树碱将被用于探测 TOPO I-DNA相互作用和4‘-(9-吖啶胺)-甲烷 间苯磺酸(MAMSA)与表鬼臼毒素VP-16 而Vm-26将用于证明Topo II与DNA的相互作用。这些 研究应该阐明参与其中的一些细节 Topo I和Topo II在细胞的复制和表达中的作用 线粒体基因组。 拓扑异构酶活性的调控具有普遍意义 但正如提案中详细说明的那样，适当的维护 染色体和染色体外DNA的超螺旋是 在癌症发生、突变和肿瘤发生中的重要作用 因此，这些研究与癌症病因学中的问题有关 和心理治疗。

The long-term objectives of my research are to understand the detailed mechanisms of mitochondrial biogenesis at the level of enzyme structure and function as well as at the level of mtDNA organization and expression. The specific goal of this proposal is to begin to elucidate the roles of DNA topoisomerases in the replication and metabolism of mtDNA. In particular, the interaction of ATP with nuclear and mitochondrial type I topoisomerase will be investigated. It has been shown that the ATP-independent type I topoisomerase is actually inhibited by ATP. Since this effect has been observed with topos from human leukemia, HeLA and calf thymus cells, we hypothesize that this ATP regulation of topo I activity may be a general phenomenon in mammalian cells. The details of the nucleotide-enzyme interaction as well as the mechanism of this regulation using in vivo and in vitro studies will be elucidated. The role of these enzymes in mitochondrial DNA replication and metabolism will be defined by investigating the interactions of topoisomerase I and II with the mitochondrial genome in vivo and in vitro. These studies will utilize several antitumor drugs which have been shown to specifically interfere with the topoisomerase- catalyzed strand breaking and rejoining process such that, in the presence of a protein denaturant, DNA cleavage results with topoisomerase covalently attached to the end of the DNA fragment. In particular, camptothecin will be used to probe for topo I-DNA interactions, and 4'- (9-acridinylamin)-methane sulfon-m-anisidide (mAMSA) and the epipodophyllotoxins VP-16 and VM-26 will be used to prove topo II-DNA interactions. These studies should elucidate some of the details of the involvement of topo I and topo II in replication and expression of the mitochondrial genome. Regulation of topoisomerase activity is of general significance but as detailed in the proposal, the maintenance of proper chromosomal and extrachromosomal DNA superhelicity is important in carcinogenesis, mutagenesis and tumorigenesis and as such these studies are relevant to problems in cancer etiology and therapy.